Revista Cienﬁca, FCV-LUZ / Vol. XXXV Recibido: 26/10/2025 Aceptado: 02/02/2026 Publicado: 26/02/2026 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 6 Revista Cienﬁca, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Field evaluaon of passive transfer in Holstein calves: Serum brix refractometry versus radial Immunodiﬀusion and associaons with early body weight change Evaluación de campo de la transferencia pasiva en terneros Holstein: refractometría de Brix en suero frente a inmunodifusión radial y asociaciones con cambios tempranos en el peso corporal. Halime Kara 1 , Mustafa Güven 2 * , Yasin Şenel 3 , Ufuk Kaya 4 ¹Department of Veterinary Medicine, Health Services Vocaonal School, Ankara Yıldırım Beyazıt University, Ankara, Türkiye ²Department of Veterinary Medicine, Menemen Vocaonal School, İzmir Bakırçay University, İzmir, Türkiye ³Kırıkkale University, Faculty of Veterinary Medicine, Department of Internal Medicine, Kırıkkale, Türkiye ⁴Department of Biostascs, Faculty of Veterinary Medicine, Hatay Mustafa Kemal University, Hatay, Türkiye *Corresponding author: mustafa.guven@bakircay.edu.tr ABSTRACT This study compared the performance of the Brix refractometer and radial immunodiﬀusion for assessing passive transfer immunity in Holstein calves and examined the relaonship between passive transfer immunity and early body weight gain during the ﬁrst 72 hours of life. A total of 136 Holstein calves that received ≥ 3 L of high-quality colostrum (Brix ≥ 22 %) within 30 minutes aﬅer birth were included. Blood samples for immunoglobulin G and Brix readings were collected 36–48 hours post-birth, and body weight was recorded at 0, 24, 48, and 72 hours. Two immunoglobulin G thresholds were applied: 24 g/L (‘excellent’ passive transfer immunity; Group 24) and 18 g/L (‘adequate’ passive transfer immunity; Group 18). Correlaon analyses were performed between radial immunodiﬀusion and Brix values, as well as between immunoglobulin G and body weight gain indices across intervals (0–24, 0–48, 0–72, 24–48, 24–72, 48–72 hours). Signiﬁcant correlaons were found between Brix and radial immunodiﬀusion (r = 0.804; P < 0.001), conﬁrming the reliability of the refractometer for ﬁeld use. Mean immunoglobulin G concentraons diﬀered signiﬁcantly between low and high groups for both thresholds (P < 0.001). No signiﬁcant immunoglobulin G diﬀerences were observed by gender (29.54 ± 1.26 vs. 28.04 ± 1.17 g/L; P = 0.432) or birth weight (≤ 38.7 vs. > 38.7 kg; P = 0.466). Correlaons between immunoglobulin G and body weight gain were weak and nonsigniﬁcant (P > 0.05). body weight gain did not diﬀer by passive transfer immunity category, although males showed slightly greater weight loss at 24–48 hours (P = 0.026) and higher gain at 48–72 hours (P = 0.048). Overall, the Brix refractometer strongly correlated with radial immunodiﬀusion, validang its praccal use, but early body weight gain was not a reliable indicator of passive transfer immunity status. Key words: Passive transfer immunity, immunoglobulin G, brix refractometer, radial immunodiﬀusion, early body weight gain. RESUMEN Este estudio comparó el rendimiento del refractómetro Brix y la inmunodifusión radial para evaluar la inmunidad de transferencia pasiva en terneros Holstein y examinó la relación entre la inmunidad de transferencia pasiva y el aumento de peso corporal temprano durante las primeras 72 horas de vida. Se incluyeron un total de 136 terneros Holstein que recibieron ≥ 3 L de calostro de alta calidad (Brix ≥ 22 %) en los 30 minutos posteriores al nacimiento. Se tomaron muestras de sangre para determinar los niveles de Inmunoglobulina G y Brix entre 36 y 48 horas después del nacimiento, y se registró el peso corporal a las 0, 24, 48 y 72 horas. Se aplicaron dos umbrales de Inmunoglobulina G: 24 g/L (inmunidad de transferencia pasiva «excelente»; grupo 24) y 18 g/L (inmunidad de transferencia pasiva «adecuado»; grupo 18). Se realizaron análisis de correlación entre los valores de inmunodifusión radial y Brix, así como entre los índices de Inmunoglobulina G y peso corporal temprano en los disntos intervalos (0-24, 0-48, 0-72, 24-48, 24- 72 y 48-72 horas). Se encontraron correlaciones signiﬁcavas entre Brix y inmunodifusión radial (r = 0,804; P < 0,001), lo que conﬁrma la ﬁabilidad del refractómetro para su uso en el campo. Las concentraciones medias de Inmunoglobulina G diﬁrieron signiﬁcavamente entre los grupos bajo y alto para ambos umbrales (P < 0,001). No se observaron diferencias signiﬁcavas en Inmunoglobulina G por sexo (29,54 ± 1,26 frente a 28,04 ± 1,17 g/L; P = 0,432) o peso al nacer (≤ 38,7 frente a > 38,7 kg; P = 0,466). Las correlaciones entre IgG y peso corporal temprano fueron débiles y no signiﬁcavas (P > 0,05). El peso corporal temprano no diﬁrió según la categoría de inmunidad de transferencia pasiva, aunque los machos mostraron una pérdida de peso ligeramente mayor a las 24- 48 horas (P = 0,026) y una ganancia mayor a las 48-72 horas (P = 0,048). En general, el refractómetro Brix se correlacionó fuertemente con el inmunodifusión radial, lo que valida su uso prácco, pero el peso corporal temprano no fue un indicador ﬁable del estado de inmunidad de transferencia pasiva. Palabras clave: Inmunidad por transferencia pasiva, inmunoglobulina G, refractómetro Brix, inmunodifusión radial, ganancia de peso corporal temprana. https://doi.org/10.52973/rcfcv-e362838

Revista Cienﬁca, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Newborn calves are born agammaglobulinaemic because the placenta of ruminant animals prevents the passage of immunoglobulins (Ig) through the placenta [1]. Within the ﬁrst hours (h) aﬅer birth, high-quality colostrum must be administered in suﬃcient quanes and at the right me, followed by its absorpon in the intesnes, to ensure passive immune protecon. The immunity thus provided is called passive transfer immunity (PTI) [2]. The failure of this process (failure of passive transfer, FPT) connues to be alarmingly prevalent worldwide. Calves experiencing FPT are more likely to develop diarrhoea and pneumonia before weaning, have a higher risk of death, and exhibit slower average body weight gain (BWG), all of which result in measurable signiﬁcant economic losses for producers [3]. The reference method used to determine PTI is the measurement of serum immunoglobulin G (IgG) levels by radial immunodiﬀusion (RID). However, RID analyses are expensive and require 24-h incubaon of samples. Therefore, portable digital or opcal Brix refractometers that determine serum total protein (STP) are preferred on farms. STP measured in the ﬁrst days of life shows a strong correlaon with serum IgG (r ≈ 0.80), but blood collecon, equipment costs, and variable cut-oﬀ values sll limit its widespread use [4 , 5]. Early BWG changes may provide a non-invasive, labour- eﬃcient alternave screening tool. Experimental studies have shown that milk intake and physical acvity during the ﬁrst ﬁve days of life predict calves’ subsequent growth and health performance, suggesng that very early weight change may reﬂect both nutrional adequacy and overall vitality. However, there appears to be insuﬃcient research in this area [6]. This study aims to invesgate the potenal use of early-stage body weight (BW) change as a non-invasive and labour-eﬃcient alternave screening method for assessing passive transfer success in Holstein calves. The current literature indicates that milk intake and physical acvity levels during the ﬁrst ﬁve days of life play a decisive role in calves’ subsequent growth performance and health status [6]. These ﬁndings support the idea that body weight change during the ﬁrst week of life can be used as both an indirect indicator of colostrum intake and nutrional adequacy and an early marker of overall vitality and health. However, there are insuﬃcient studies validang early weight change as a screening tool in relaon to passive immunity status in calves [6]. This research aims to ﬁll this gap in literature. MATERIALS AND METHODS Ethical approval The study protocol was conducted with the approval of the Ankara University Local Ethics Commiee for Animal Experiments, dated 07.08.2024 and numbered 2024-12-95. Study design and animal material This study was conducted on healthy Holstein calves born between January and July 2025 at the same dairy farm. A total of 136 calves were included in the study, whose umbilical cords were disinfected with povidone-iodine (7 %) within ≤ 2 h of birth and who were fed ≥ 3 L of high-quality colostrum (Brix ≥ 22 %) within the ﬁrst 30 minutes (min). Calves that were sllborn, aborted, died within the ﬁrst 72 h, had diarrhoea or pneumonia, were weak and unable to stand on their own, had no suckling reﬂex, or were born by caesarean secon or diﬃcult delivery were excluded from the study. Group formaon The calves, whose passive transfer immunity levels were determined by serum Brix refractometer (Milwaukee, MA871, USA), were divided into two groups: low and high. The BW changes at 24, 48, and 72 h, the eﬀects of gender, and the serum IgG levels of the groups determined by the Brix refractometer were compared. To assess the passive immunity status of calves, blood was collected via a single jugular vein puncture at 36-48 h of age. At the same me, BW was measured at birth (0 h) and at 24, 48, and 72 h using an electronic scale with ± 0.1 kg accuracy (Kontar, 75-125cm/ 600 kg, Türkiye). When forming the groups, a preliminary assessment using a Brix refractometer determined that there were too many animals above the passive immunity transfer cut-oﬀ value due to environmental and herd management success at the farm where the samples were collected. In order to group the samples, calf sera with high and low Brix values were subjected to RID analysis using the Brix value as a reference, and groups were formed based on the IgG levels obtained from the RID results. Two diﬀerent groupings were made using threshold values of 18 g/L and 24 g/L, with values below the threshold forming the low group and values above the threshold forming the high group. Laboratory analyses Fresh serum samples were analysed using a portable digital refractometer (Milwaukee MA871, USA; measurement range 0–30 % Brix). Passive transfer success was deﬁned as a cut-oﬀ value ≥ Brix %7.8 [7]. The IgG level in the same samples was measured using a ra- dial immunodiﬀusion kit (JJJ Diagnosc, Kent Laboratories Inc., USA) [8]. An IgG value < 10 g/L was used as the threshold value for passive transfer insuﬃciency (PTI), an IgG level ≥ 18 g/L was used as a reference for adequate PTI, and a level ≥ 24 g/L was used as a reference for excellent PTI [9]. Stascal analysis In the study, the Shapiro–Wilk normality test was ﬁrst applied to evaluate the distribuon characteriscs of the data for stascal evaluaon. For intergroup comparisons, the independent samples t-test was used for parameters showing a normal distribuon, and the Mann–Whitney U test was used for data not showing a normal distribuon. The interacon between the BW group and sex on the % Brix value, IgG levels, 2 of 6

Field evaluaon of passive transfer in Holstein calves / Kara et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico and weight variables was examined using two-way analysis of variance (ANOVA). In correlaon analyses, the relaonships between BW changes, % Brix value, and IgG levels were calculated using Pearson or Spearman correlaon coeﬃcients. All analyses were performed using IBM SPSS Stascs version 23.0, with an alpha level set at 0.05. Given the limitaons of the study, the research was conducted on a single farm, with a limited sample size and a short follow-up period. This limits the external validity and the capacity to evaluate long-term results. Although measurements were taken using electronic scales, variables such as weighing me, digesve tract content, and hydraon status may aﬀect the accuracy of weight data. Future studies should deepen the subject with a mul-farm design, weekly growth monitoring, and a holisc approach that includes indicators of immunity, metabolism, and gut health. RESULTS AND DISCUSSION In this study, two main ﬁndings regarding PTI measurement in Holstein calves within the ﬁrst 72 h aﬅer birth stand out. The correlaon between RID-IgG measurement using a Brix refractometer in ﬁeld condions is strong, and although the thresholds reported as 18 g/L for ‘adequate’ and 24 g/L for ‘excellent’ IgG clearly disnguish the groups, they do not show a signiﬁcant correlaon with early body weight changes. These results do not allow the use of early-stage body weight changes to monitor passive transfer success. A high level of stascally signiﬁcant correlaon was found between serum refractometer measurement and IgG level (r = 0.804; P < 0.001, TABLE I). TABLE I Correlaon between refractometer and IgG Refractometer IgG Refractometer 1 0.804 *** IgG 1 ***: P<0,001 The Pearson correlaon coeﬃcient between Brix refractometer (%) measurements and serum immunoglobulin G (IgG; g/L) concentraons determined by the radial immunodiﬀusion (RID) method. IgG: Immunoglobulin G. When examining refractometer–IgG agreement and clinical relevance, the high correlaon idenﬁed between the refractometer and IgG (r = 0.804; P < 0.001) is consistent with reports in the literature. Deelen et al. [10] reported a correlaon of r ≈ 0.93 between % Brix and RID-IgG in 397 calf sera and shared that the sensivity-speciﬁcity balance was best at an 8.4 % threshold for % Brix. Similarly, systemac reviews emphasise that refractometer-based tests demonstrate sasfactory accuracy in detecng PTI for < 10 g/L IgG and are advantageous for herd PTI monitoring due to their operaonal applicability. Findings of this study support that while IgG remains the gold standard, refractometers can also be used as a valid criterion in the ﬁeld [9 , 10 , 11 , 12]. Despite a strong correlaon being established between IgG and Brix refractometer, the failure of preliminary assessments using the Brix refractometer to successfully predict the 10 g/L threshold indicates that the predicon success rate for IgG thresholds below 10 and 18 g/L using the refractometer is moderate, while the predicon success rate for IgG thresholds above 25 g/L is high Akköse et al. [13] report that the refractometer’s predicon success for IgG thresholds below 10 and 18 g/L was moderate, while predicon success for IgG thresholds above 25 g/L was quite good. Immunoglobulin G means were expectedly high for both threshold values, and a signiﬁcant diﬀerence was observed between the high and low groups (TABLES II). In the IgG measurement, the mean IgG measurement results in the low category at the ≥ 24 g/L threshold value were signiﬁcantly lower than in the high category (20.70 ± 0.46 vs 35.98 ± 0.91 g/L; P < 0.001, TABLE II). A similar diﬀerenaon was observed at the ≥ 18 g/L threshold (16.00 ± 0.50 vs 31.09 ± 0.83 g/L; P < 0.001, TABLE II). TABLE II Serum immunoglobulin G concentraons in calves classiﬁed according to 24 g/L and 18 g/L thresholds Parameter Group (24) P Group (18) P Low High Low High IgG (g/L) 20.70 ± 0.46 35.98 ± 0.91 < 0.001 16.00 ± 0.50 31.09 ± 0.83 < 0.001 Data are presented as mean ± standard error. Serum immunoglobulin G (IgG) concentraons were measured by the radial immunodiﬀusion (RID) method. Calves were classiﬁed into Low (< cut-oﬀ) and High (≥ cut-oﬀ) groups based on 24 g/L and 18 g/L thresholds. Comparisons between groups were analysed using the independent samples t-test. In this study, the low and high groups were stascally signiﬁcantly diﬀerenated for both thresholds of 18 and 24 g/L, which were determined as IgG cut-oﬀ values. This is consistent with the mul-level PTI classiﬁcaon proposed by Lombard et al. [9] (excellent ≥ 24, good 18–24.9, moderate 10–17.9, poor < 10 g/L) [9 , 14]. Correlaons between serum IgG levels determined by RID and BW changes during the ﬁrst 72 h were not stascally signiﬁcant (all P > 0.05; TABLE III). There was a posive correlaon between IgG levels and body weight levels, but this was not stascally signiﬁcant. TABLE III Correlaons between immunoglobulin G and body weight indices IgG BW 0-24 BW 0-48 BW 0-72 BW 24-48 BW 24-72 BW 48-72 IgG r 1 0.056 0.062 0.090 0.021 0.073 0.051 P 0.522 0.479 0.300 0.807 0.401 0.558 Pearson correlaon coeﬃcients examining the relaonships between serum immunoglobulin G (IgG; g/L) concentraons and body weight (BW) gain indices across diﬀerent me intervals. 3 of 6

Revista Cienﬁca, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Although BWG were higher in the group with blood IgG levels below 18 g/L in calves during the ﬁrst 72 h, the diﬀerence was not stascally signiﬁcant (TABLE IV). Although BWG were higher in the ﬁrst 72 h in the group of calves with blood IgG levels below 24 g/L, the diﬀerence was not stascally signiﬁcant (TABLE IV). TABLE IV Body weight indices (kg) in calves classiﬁed according to 18 g/L and 24 g/L immunoglobulin G thresholds Parameter Threshold (18 g/L) P Threshold (24 g/L) P Low High Low High BW24-0 2.07 ± 0.74 1.11 ± 0.21 0.101 1.44 ± 0.33 1.10 ± 0.26 0.410 BW48-0 1.75 ± 0.77 0.91 ± 0.22 0.183 1.26 ± 0.34 0.84 ± 0.30 0.359 BW72-0 2.79 ± 0.71 1.82 ± 0.21 0.099 2.17 ± 0.32 1.78 ± 0.27 0.354 BW48-24 -0.32 ± 0.23 -0.20 ± 0.11 0.674 -0.18 ± 0.12 -0.25 ± 0.16 0.742 BW72-24 0.72 ± 0.22 0.71 ± 0.11 0.982 0.73 ± 0.16 0.69 ± 0.13 0.823 BW72-48 1.04 ± 0.16 0.91 ± 0.11 0.645 0.92 ± 0.12 0.94 ± 0.15 0.905 Data are presented as mean ± standard error. Body weight (BW) gain indices were calculated for calves categorized according to 18 g/L and 24 g/L serum immunoglobulin G (IgG) thresholds. IgG concentraons were measured by the radial immunodiﬀusion (RID) method. Groups (Low vs. High) were compared using the independent samples t-test When the relaonship between early-stage BW changes and passive immunity was observed, no signiﬁcant correlaon was found between IgG measurement results and 0–72-h BW gain indices. Mila et al. [15] reported in a study conducted on dogs that they found a signiﬁcant result aﬀecng passive transfer success between daily BW change on the second day aﬅer birth and serum IgG level [15]. Furthermore, it was considered that weighing ming, hydraon status, and the amount of content in the digesve tract may have contributed to the lack of stascally signiﬁcant diﬀerences in BWG between groups in the 18 and 24 g/L classiﬁcaons. Therefore, the weight change dynamics in the short term also have limited ability to reﬂect the biological eﬀect of PTI. The literature indicates that the eﬀect of PTI on clinical outcomes is more evident in the medium to long term, such as reduced morbidity and mortality, daily BWG, and advanced performance. In this context, comprehensive reviews and meta- analyses demonstrang the protecve eﬀect of strong PTI on neonatal health report a signiﬁcant increase in the risk of death and disease in PTI. The ﬁnding that the study results indicated a signiﬁcant weight diﬀerence between groups in the short-term follow-up is consistent with exisng evidence demonstrang that the beneﬁts of PTI become more apparent in the long term [14 , 16]. In the evaluaon, two groups with birth weights below and above 38.7 kg were compared, and no stascally signiﬁcant diﬀerence was observed between the groups (TABLE V). The serum IgG level of female calves aged 36-48 h was measured as 29.54 ± 1.26 g/L, while that of male calves was measured as 28.04 ± 1.17 g/L. No stascally signiﬁcant diﬀerence was observed between the serum IgG levels of female and male calves (TABLE V). TABLE V Values immunoglobulin G g/L according to Birth Weight and Sex to calves Parameter Birth Weight (kg) P Sex P ≤ 38.7 > 38.7 Female Male IgG (g/L) 29.46 ± 1.04 28.19 ± 1.44 0.466 29.54 ± 1.26 28.04 ± 1.17 0.432 Data are presented as mean ± standard error. Serum immunoglobulin G (IgG) concentraons were measured by the radial immunodiﬀusion (RID) method. The eﬀects of birth weight (classiﬁed by 38.7 kg threshold) and sex on IgG levels were analysed using the independent samples t-test. The ﬁndings, indicang that serum IgG levels did not diﬀer signiﬁcantly according to sex and birth weight, suggest that under opmal condions where management procedures are standardized, colostrum management, genecs, and sex—as inherent factors inﬂuencing passive transfer adequacy are not primary determinants. Rather, the provision of appropriate colostrum remains of crical importance. Indeed, Godden et al. [14] and Weaver et al. [17] also emphasize that providing an adequate quanty of high-quality colostrum as soon as possible aﬅer birth is the most fundamental determinant of passive transfer success. Body weight indices were similar between the two me periods in terms of sex, although a signiﬁcant diﬀerence was observed between the two me periods (TABLE VI). BW showed a more pronounced short-term weight loss in males compared to females in the 24-48 range (−0.57 ± 0.22 vs −0.06 ± 0.12 kg; P = 0.026), while a higher BW increase was observed in males in the subsequent BWG 48-72 range (1.23 ± 0.18 vs 0.78 ± 0.13 kg; P = 0.048). No stascally signiﬁcant gender-related diﬀerences were observed in the other indices. TABLE VI Body weight indices (kg) according to the sex Parameter Group (IgG 24) P Female Male BW24-0 1.20 ± 0.24 1.31 ± 0.48 0.820 BW48-0 1.14 ± 0.23 0.74 ± 0.54 0.496 BW72-0 1.92 ± 0.23 1.96 ± 0.49 0.934 BW48-24 -0.06 ± 0.12 -0.57 ± 0.22 0.026 BW72-24 0.72 ± 0.12 0.65 ± 0.19 0.754 BW72-48 0.78 ± 0.13 1.23 ± 0.18 0.048 Body weight (BW) gain indices (kg) in calves classiﬁed according to female and male calves, compared using the independent samples t-test to evaluate the eﬀect of sex across diﬀerent postnatal me intervals. While studies in the literature report higher IgG concentraons in female calves compared to male calves [18], larger sample studies have reported no signiﬁcant diﬀerence 4 of 6

Field evaluaon of passive transfer in Holstein calves / Kara et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico between the sexes [19]. In the present study, the body weight diﬀerence observed in the early period (decrease between 24-48 h, recovery at 72 h), which was stascally signiﬁcant, may be due to temporary variaon, considering the relavely short follow-up period, limited sample size, possibility of measurement errors, and physiological ﬂuid balance changes speciﬁc to the early postnatal period in calves. It is also thought that weight change in the ﬁrst 72 h may be strongly inﬂuenced by factors that ﬂuctuate rapidly, such as placental ﬂuid loss at birth, thermoregulaon-related eﬃciency loss, and individual diﬀerences in colostrum intake rate [20 , 21]. The use of IgG as a reference method and the collecon of blood samples at 36-48 h is consistent with the current literature in the assessment of PTI [22 , 23]. At the same me, data obtained from a single farm reduced potenal variaons due to homogeneous management but limited the diversity of management procedures. The short follow-up period of 72 h may be insuﬃcient to understand the cumulave eﬀects of PTI or PTI on growth and health [9 , 12]. IgG and refractometer measurements can be used complementarily in the ﬁeld, with IgG-based thresholds of 18 and 24 g/L reliably disnguishing calves, but PTI does not show a linear and immediate relaonship with early (0–72 h) weight changes. The ﬁndings support that the diagnosc focus in PTI assessment should be on serological measurements and colostrum management processes rather than short-term body weight change dynamics [9 , 10]. CONCLUSION As a result of this study, it is recommended that refractometer- based Brix measurements connue to be used in PTI screenings. Importantly, adequate colostrum consumpon remains the cornerstone of successful passive transfer; therefore, ensuring mely intake of a suﬃcient volume of high-quality colostrum and maintaining proper colostrum management pracces should be emphasized as a primary prevenve strategy against PTI failure under ﬁeld condions. However, body weight monitoring should be considered as a complementary metric for long-term growth performance and health monitoring rather than as a short-term PTI indicator. It is thought that future studies should connue to invesgate fast, low-cost, and non-invasive strategies for PTI screening under ﬁeld condions. Conﬂict of interest statement The authors declare that there is no conﬂict of interest. 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